The resolution in a fluorescence microscope is given by the point spread function (PSF). In wide field microscope laterally extended structures are axially not resolved because of the missing cone in the optical transfer function (OTF). Further more out of focus structures blur the image of structures in the focus.
In 2-photon fluorescence microscopy, the fluorescence of the fluorophore in the sample is only excited by two photons of the optical excitation signal each providing a half of the necessary excitation energy. As a result, the PSF of the excitation of the fluorophore is depending on the square of the intensity distribution or of the PSF of the optical excitation signal. As compared to the PSF of the optical excitation signal, the square of the PSF of the optical excitation signal is more concentrated towards the centre of the excitation-volume in all directions, particularly including the axial direction in which the optical excitation signal is focussed into the sample. In this way the spatial resolution in axial direction is directly increased by 2-photon excitation of the fluorophore.
A further known measure to increase the axial resolution in fluorescence microscopy is confocal detection of the fluorescence light which means a spatial limitation of the detection volume out of which the fluorescence light is actually detected. Here, the effective PSF is the product of the PSF of the optical excitation signal and of the PSF of the detection arrangement. 2-photon excitation may also be combined with confocal detection.
In more elaborate approaches, the PSF of the optical excitation signal is spatially confined by means of superimposing coherent parts of the optical excitation signal for forming an interference pattern, the fluorophore substantially only being excited at the maxima of the interference pattern. A prominent type of this kind of fluorescence microscopy is 4Pi microscopy in which spherical wave fronts of the excitation signal are superimposed from two opposite directions in a common focal spot for forming an interference pattern. Within the focal spot, however, not only the first order maximum of the interference pattern is found at the centre but second order maxima flank this first order maximum on both sides of the focal plane. To make use of the full potential of 4Pi microscopy, the second order maxima have to be suppressed in some way. One option is 2-photon excitation as discussed above. Another option is coherent detection of the fluorescence light both providing a further factor of the total PSF.
A further approach of suppressing the fluorescence light from the second order maxima in 4Pi microscopy is forcing the fluorophore within the area of the second order maxima to stimulated emission of fluorescence light which is discriminated from the detected fluorescence light from the sample. Generally, in stimulated emission depletion (STED) fluorescence microscopy an excitation-volume in which fluorophore molecules have been excited for fluorescence is confined or reduced in dimensions by de-exciting a part of the fluorophore molecules within the excitation-volume, only leaving the fluorophore molecules within a strongly confined centre of the excitation-volume in their fluorescent state so that the registered fluorescence light can be assigned to the fluorophore molecules within a very small effective excitation-volume.
For a survey of the above methods of fluorescence microscopy see Matthias Nagorni and Stefan W. Hell “Coherent use of opposing lenses for axial resolution increase in fluorescent microscopy. I. Comparative Study of concepts” in J. Opt. Soc. Am. A/Vol. 18, No. 1/January 2001, p. 36-48.
The basic principle known from STED fluorescence microscopy is also applied in reversible saturable optical fluorescence transition (RESOLFT) microscopy in which photochromic fluorophores are used to confine the effective excitation-volume in which an optical excitation signal excites a fluorophore for fluorescence. To this end, a switching signal by which the photochromic fluorophore can be switched in an off or dark state and which has a zero point at the centre of the excitation-volume is also applied to the sample. If the zero point is a zero point of an interference pattern, and if the intensity of the switching signal is sufficiently high, the dimensions of the effective excitation-volume in which the fluorophore is still in its on or fluorescent state and from which the detected fluorescence light can exclusively origin may be kept far below the diffraction barrier (see, for example, U.S. Pat. No. 7,064,824).
The concept of switchable photochromic fluorophores is also used in photo-activated localization microscopy (PALM) (see WO 2006/127692 A2) and stochastic optical reconstruction microscopy (STORM). Here, such a small proportion of a total concentration of a photochromic fluorophore is switched on into its fluorescent state that the fluorescence light from the single fluorescent fluorophore molecules can be registered separately, and Gaussians are fitted to the PSF of the spatial distribution of the registered fluorescence light to determine the actual location of the fluorescent molecule with sub diffraction spatial resolution. When the few switched on fluorophore molecules photobleach, another subset of the fluorophore molecules is switched into the on state and the procedure is thus repeated for sufficient stochastically selected subsets of the fluorophore molecules to image a structure labelled with the fluorophore.
A strong disadvantage of PALM and STORM is the time consumed by the procedure which limits the observation of living samples, i.e. of objects in which the structure of interest changes with time, and the production of images which may be directly viewed with the human eye.
RESOLFT and STED microscopy as well as 4Pi microscopy and other similar approaches to high spatial three-dimensional resolution fluorescence microscopy also require sophisticated equipment.
2-Photon excitation of fluorophores leads to a 3-dimensional resolution without additional means but the fluorescence yield is comparably low. Additionally a large difference in the wavelengths of the excitation light and the fluorescence light has to be coped with in 2-photon fluorescence microscopy. In confocal laser scanners chromatic aberrations may also be limiting for the precision of the object registration. Here often some axial resolution has to be sacrificed to achieve a large enough signal strength by opening the detection pinhole which additionally requires precise optical alignment.
Ando et al. (2007, see “References”) disclose a method of imaging a structure in a sample labelled with the photochromic fluorophore Dronpa. Via a common objective the sample is subjected to irradiation in a common focal spot both at 405 nm and at 488 nm, and fluorescence light emitted by the fluorophore is confocally detected. The irradiation at 405 nm transfers Dronpa out of a dark or non-fluorescent state into a fluorescence state. The irradiation at 488 nm excites Dronpa for emission of fluorescence light, and returns it into its dark or non-fluorescence state. Whereas the irradiation at either 405 nm or 488 nm alone produces no fluorescence light signal, the simultaneous irradiation at both wavelengths produces a fluorescence light signal. Confocal detection of this fluorescence light results in a spatial resolution typical for fluorescence microscopy with confocal detection. Ando et al. propose that it should be possible to spatially restrict the generated fluorescence signal, which can be collected in wide-field detection mode, if the two lasers supplying the irradiation light of the two different wavelengths are aligned independently using separate objectives.
A method of fluorescence microscopically imaging a structure in a sample with high 3-dimensional spatial resolution which provides additional spatial resolution as compared to known methods at little additional efforts would be highly appreciated.